We wish to pursue the molecular mechanism(s) by which 25-hydroxycholesterol, which is oxygenated sterol analog of low density lipoprotein-bound cholesterol, activates the activity of the microsomal enzyme fatty acyl coenzyme A: cholesterol acyltransferase (ACAT) in Chinese hamster ovary (CHO) cells. Our planned experimentation is summarized as follows: (i) Extensively purify solubilized ACAT pig liver microsomal fraction. (ii) Reconstitute the purified ACAT fraction into well defined single-wall phospholipid-cholesterol vesicles. Characterize the vesicles by different physical criteria. (iii) Solubilize and reconstitute the microsomal ACAT prepared from CHO cell extracts. (iv) Use the solubilization-reconstitution procedure as a standard enzyme assay to determine the mechanism(s) of the ACAT activation process by various sterols or sterol analogs in CHO cells. (v) Use the purified ACAT fraction from pig liver microsome and various soluble or particulate factors present in CHO cell homogenate to generate the ACAT activation in vitro. Isolate and characterize the components necessary for activation to occur. This research project requires expertises in enzymology and cell biology. The outcome of this research project may help our understanding of the moleculr mechanism(s) of cholesterol ester accumulation in human atherosclerotic plaques.